Abstract Microhomology-mediated end joining (MMEJ) is an evolutionary conserved pathway to repair DNA double strand breaks (DSBs) by annealing small stretches (2-20 bps) of overlapping sequence (microhomology; MH) flanking the break site. By design, MMEJ is highly mutagenic because it always results in the deletion of one of the MH and the inter-MH sequences. MMEJ also frequently leads to chromosomal rearrangements due to its ability to engage in promiscuous end joining. Moreover, we found that MMEJ is hypermutagenic, and accumulates mutations flanking the repair junctions up to several kilobases from the break. Despite these risks, MMEJ is widely adopted in cells as an alternative option to high fidelity DSB repair for a host of biological events including copy number variations, immune system development, and telomere fusions. Most recently, MMEJ has also been implicated in pathogenic chromosomal translocations and is emerging as a promising therapeutic target in several types of cancers. Together these observations underscore the importance of MMEJ as a genome destabilizer and warrant further studies to define genetic and biochemical mechanisms of MMEJ and its regulation under various environmental and metabolic conditions. The purpose of this application is to resolve several fundamental questions on the physiological and pathological roles of MMEJ common to all eukaryotic cells. One of the key unresolved questions in MMEJ is how cells recognize and select specific MHs for annealing and the effect of position and the number of mismatches in MH on this process. We also do not know what are the factors catalyzing MH annealing and how cells regulate repair choice to limit unwanted MMEJ and suppress repair-associated chromosomal instability. Lastly, we will examine why aging triggers changes in repair pathway choice and how MMEJ activity contributes to age related chromosomal instability in yeast cells. Our preliminary results already unraveled several interesting rules and parameters in MH annealing and identified end processing factors that are likely involved in this reaction. We also developed a novel reporter to monitor repair choice that for the first time includes MMEJ as one of the DSB repair options, and to evaluate the roles of DNA damage response and chromatin remodelers in the decision making process. The outcomes of this investigation will reveal the underlying mechanism of key steps in MMEJ and functional interactions between DSB repair pathways to sustain chromosomal integrity in response to various environmental and physiological cues. The results will also help establish precise contributions of MMEJ in many genome modification events and predict the frequency and types of MMEJ products at random sequences in gene editing and at-risk genome sequences.